The detection of hydrolytic enzymes has been extensively used in diagnostic assays ranging from immunoassays, nucleic acid assays, receptor assays, and other assays, primarily due to their high sensitivity and non-radioactivity. The hydrolytic enzymes include phosphatases, glycosidases, peptidases, proteases, and esterases. By far, most commonly used are phosphatases and glycosidases. For instance, alkaline phosphatase has been extensively used as a label in various enzyme-linked immunosorbent assays (ELISAs) due to its high turn-over rate, excellent thermal stability and ease of use. Many glycosidases, such as β-galactosidase and β-glucuronidase, have also been used in ELISA due to their very high selectivity for the hydrolysis of their preferred substrates. On the other hand, some hydrolytic enzymes play important functions by themselves in biological processes of the human body and microorganisms. Therefore, direct detection of these markers is another important aspect of diagnostics.
In connection with the detection of hydrolytic enzymes, there are three types of substrates: chromogenic, fluorogenic and chemiluminescent substrates. Among them, chemiluminescent substrates offer the best enzyme detection sensitivity due to the intrinsic advantages of higher detectability of chemiluminescent product, or lower substrate and instrumental backgrounds, and less interference from biological samples. Therefore, there has been a steady trend towards developing chemiluminescent substrates and applying them in a variety of diagnostics.
Stable Dioxetanes
One class of widely used chemiluminescent substrates for hydrolytic enzymes are stable dioxetanes (Bronstein et al., U.S. Pat. Nos. 4,931,223, 5,112,960, 5,145,772 and 5,326,882; Schaap et al., U.S. Pat. Nos. 5,892,064, 4,959,182 and 5,004,565; and Akhavan-Tafti et al., U.S. Pat. No. 5,721,370). Here, the thermally stable protective group on the phenolic moiety of the dioxetane substrates is cleaved by a hydrolytic enzyme of interest, such as alkaline phosphatase (AP) or β-galactosidase, depending on whether the protective group is a phosphoryl or β-D-galactopyranosidyl group. The newly generated dioxetane phenoxide anion undergoes auto-decomposition to a methoxycarbonylphenoxide in an electronically excited state. The latter then emits light at λmax˜470 nm.
In an aqueous environment where virtually all biological assays are performed, the decomposition of the dioxetanes produces chemiluminescence in a very low quantum yield, typically about 0.01%, and a slow kinetics with t1/2 1˜10 minutes. This is quite different from the decomposition of the dioxetanes in an organic environment. For instance, the dioxetane having the phenol moiety protected by an acetyl group or a silyl group, upon treatment with a base or fluoride, exhibits quantum yield up to 25% in DMSO and 9.4% in acetonitrile, respectively, and t1/2 is about 5 sec. at 25° C. Schaap, et al., Tetrahedron Letters, 28(11), 1155, (1987) and WO 90/07511 A.
Voyta et al., (U.S. Pat. No. 5,145,772) disclosed a method of intermolecular enhancement of quantum yield of the dioxetane products using polymeric ammonium salts, which provide a hydrophobic environment for the phenoxide produced by the enzyme.
Akhavan-Tafti et al., reported methods of intermolecular enhancement of quantum yield of the dioxetane products using polymeric phosphonium salts (U.S. Pat. No. 5,393,469) and dicationic surfactants (U.S. Pat. Nos. 5,451,347 and 5,484,556).
Schaap et al., (U.S. Pat. Nos. 4,959,182 and 5,004,565) disclosed another method for increasing quantum yield of the dioxetane products using fluorescent co-surfactants as energy acceptors. The resonance energy embodied in the excited phenoxide produced by the enzyme is effectively transferred to the fluorescent co-surfactants. Instead of emitting light at λmax 470 nm characteristic of the dioxetane, this system emits light at λmax 530 nm as a result of energy transfer to the highly efficient fluorophore, fluorescein.
Another approach for improving quantum yield of the dioxetanes, disclosed by Schaap et al., (U.S. Pat. No. 5,013,827), is to covalently attach a fluorophore having high quantum yield to the light emitting phenoxide moiety. The resonance energy from the excited phenoxide is intramolecularly transferred to the attached fluorophore. The latter in turn emits light at its own wavelength. It is claimed that such dioxetane-fluorophore conjugates exhibit quantum yield as high as 2%.
Wang et al., (WO 94/10258) unveiled a class of electron-rich, aryl-substituted dioxetane compounds in which the aryl group is poly-substituted with a suitable electron-donating group so that intense luminescence is observed.
Akhavan-Tafti et al., (U.S. Pat. No. 5,721,370) provided a group of stable chemiluminescent dioxetane compounds with improved water solubility and storage stability. The compounds are substituted with two or more hydrophilic groups disposed on the dioxetane structure and an additional fluorine atom or lower alkyl group.
Schaap et al., (U.S. Pat. No. 5,892,064) disclosed a class of chemiluminescent dioxetane compounds substituted on the dioxetane ring with two nonspirofused alkyl groups.
Urdea at al. (EP Application 0401001 A2) described another sub-class of dioxetane compounds that can be triggered by sequential treatment with two different activating enzymes to generate light. The system rests on the principle that the dioxetane substrates have two protecting groups that can be removed sequentially by different processes to produce an excited phenoxide, and the removal of the first protecting group is triggered by the enzyme used as a label in the assay.
Luminol Substrates
Sasamoto at al., Chem. Pharm. Bull., 38(5), 1323 (1990) and Chem. Pharm. Bull., 39(2), 411 (1991), reported that o-aminophthalhydrazide-N-acetyl-β-D-glucosaminide (Luminol-NAG) and 4′-(6′-diethylaminobenzofuranyl)-phthalhydrazide-N-acetyl-β-D-glucosaminide, both being the non-luminescent forms of luminol, are substrates of N-acetyl-β-D-glucosaminidase. Upon the action of the enzyme on these substrates, luminol or luminol derivative is generated, which then can be detected by triggering with 0.1% hydrogen peroxide and a peroxidase (POD) or Fe(III)-TCPP complex catalyst to release a chemiluminescent signal.
Enzyme-Modulated Protected Enhancer and Anti-Enhancer
Kricka (U.S. Pat. No. 5,306,621) disclosed that light intensity of certain peroxidase-catalyzed chemiluminescent reactions can be modulated by AP that acts on a pro-enhancer or a pro-anti-enhancer. For example, the intensity of a chemiluminescent reaction containing luminol, horseradish peroxide and hydrogen peroxide can be enhanced by an enhancer (4-iodophenol) liberated by the enzymatic action of AP on a pro-enhancer (4-iodophenol phosphate), thus enabling AP to be assayed. Alternatively, in the same above reaction where additional enhancer (4-iodophenol) is present, the light intensity can be decreased by an anti-enhancer (4-nitrophenol) generated by the enzymatic action of AP on a pro-anti-enhancer (4-nitrophenol phosphate). In the latter format, the presence of AP can be assayed by measuring the reduction in the light intensity.
Similar to the above, an assay using enzyme-triggerable protected enhancer for quantitation of hydrolytic enzymes was also unveiled by Akhanvan-Tafti in EP Application 0516948 A1.
Akhanvan-Tafti et al., (WO 96/07911) disclosed another method of detecting hydrolytic enzymes based on the principle of the protected enhancer, where the light emitting species is generated from the oxidation of acridan by peroxidase and peroxide.
Acridene Enol Phosphate
Akhanvan-Tafti et al., (U.S. Pat. No. 5,772,926 and WO 97/26245) disclosed a class of heterocyclic, enol phosphate compounds represented by the non-luminescent acridene enol phosphate. Upon the reaction with a phosphatase enzyme, acridene enol phosphate is converted to the dephosphoryl enolate, which reacts with molecular oxygen to produce light (see scheme below). It was also disclosed that the light output was greatly enhanced by the addition of a cationic aromatic compound to the assay system.
Other Chemiluminescent Substrates for Hydrolytic Enzymes
Vijay (U.S. Pat. No. 5,589,328) disclosed chemiluminescent assays that detect or quantify hydrolytic enzymes, such as alkaline phosphatase, that catalyze the hydrolysis of indoxyl esters. The assay includes the steps of reacting a test sample with an indoxyl ester and then immediately or within a short time (typically less than about 15 minutes) measuring the resulting chemiluminescence. The resulting chemiluminescence may be amplified by adding a chemiluminescent enhancing reagent.
Among the above chemiluminescent hydrolytic enzyme methods, the dioxetane system appears to be the most sensitive detection system and therefore has been increasingly used in various assays. Despite its widespread use, this system has an inherent drawback in that background chemiluminescence in the absence of enzyme is observed due to slow thermal decomposition and non-enzymatic hydrolysis of the dioxetane. Another intrinsic disadvantage is that the phenoxide, once generated by enzymatic reaction, is extremely unstable and readily undergoes decomposition to release light. In this sense, the phenoxide, the light emitting species, is never “accumulated” during the enzymatic reaction. Therefore, it is one of the major goals of this invention to provide new chemiluminescent substrates whose derived chemiluminescent products have distinguishable emission profiles and whose total signal can be accumulated during the enzymatic reaction, thereby providing an alternative and sensitive detection method for hydrolytic enzymes.